Pk2 inhibits hexagonal packing in the gastrula ectoderm. (A) Scheme of the experiment. Flag-Pk2 RNA was coinjected with GFP reporter RNA into two ventral blastomeres at the four- to eight-cell stage. Time-lapse imaging of the animal superficial ectoderm was initiated at stage 10. (B and C) Snapshot images of gastrula ectoderm expressing myrGFP only (B) and myrGFP with Flag-Pk2 (C) from Video 6. Left: 0 h. Right: 4 h 55 min. (D–F) Higher resolution snapshot images of ectoderms expressing myrGFP only (D) or with Flag-Pk2 (E) from Video 7. Imaging was initiated at stage 11.5. Unpigmented albino embryos were used to highlight myrGFP fluorescence signals from the lateral membrane, which are shown in red in the schematic diagram (F). (G–J) Quantification of hexagonal cell packing. Pigmented wild-type embryos were used for segmentation and quantification. Cell packing was assessed by apical domain size heterogeneity (G), AJ linearity (I), and the number of neighboring cells (J). Schematic of AJ linearity quantification (H). The actual length of the AJs is divided by the shortest distance between two vertices (L/L′). Dots represent the values of average AJ linearity in individual embryos (I). The lines correspond to the average values of three embryos. (J) Frequency distribution histogram of the number of neighboring cells. Original numerical data were used for statistical analysis in G and I. The Kolmogorov‒Smirnov test was used to compare distributions in G. One-way ANOVA was used to compare the means of three or more groups in I. The Bonferroni correction was used for pairwise multiple comparisons. The chi-square test was used in J. The total number of cells or AJs scored in G–J was as follows: 576 cells and 1,127 AJs (myrGFP control, 00:00 h); 672 cells and 1,169 AJs (myrGFP control, 4:55 h); 724 cells and 1,360 AJs (myrGFP+F-Pk2, 00:00 h); and 764 cells and 1,355 AJs (myrGFP+F-Pk2, 4:55 h). Three embryos per group were used. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. Scale bars: 50 μm (B and C) and 25 μm (D and E).
